US4352883A - Encapsulation of biological material - Google Patents

Encapsulation of biological material Download PDF

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Publication number
US4352883A
US4352883A US06/024,600 US2460079A US4352883A US 4352883 A US4352883 A US 4352883A US 2460079 A US2460079 A US 2460079A US 4352883 A US4352883 A US 4352883A
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US
United States
Prior art keywords
tissue
capsules
droplets
core material
gum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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US06/024,600
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English (en)
Inventor
Franklin Lim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Biotech Inc
Repligen Corp
Original Assignee
Damon Corp
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Application filed by Damon Corp filed Critical Damon Corp
Priority to US06/024,600 priority Critical patent/US4352883A/en
Priority to GB8008971A priority patent/GB2046209B/en
Priority to CA000348524A priority patent/CA1145258A/en
Priority to DK130580A priority patent/DK130580A/da
Priority to CH187/84A priority patent/CH657786A5/de
Priority to IT67472/80A priority patent/IT1133081B/it
Priority to SE8002357A priority patent/SE448060B/sv
Priority to FR8006875A priority patent/FR2452285B1/fr
Priority to BE0/199986A priority patent/BE882476A/fr
Priority to CH2423/80A priority patent/CH653914A5/de
Priority to DE19803012233 priority patent/DE3012233A1/de
Priority to JP3912380A priority patent/JPS55157502A/ja
Priority to NO800901A priority patent/NO160380C/no
Priority to FR8020768A priority patent/FR2457688B1/fr
Priority to US06/259,595 priority patent/US4391909A/en
Priority to US06/372,835 priority patent/US4409331A/en
Priority to GB08227739A priority patent/GB2119734B/en
Application granted granted Critical
Publication of US4352883A publication Critical patent/US4352883A/en
Priority to GB08306682A priority patent/GB2119737B/en
Assigned to DAMON BIOTECH, INC. reassignment DAMON BIOTECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: DAMON CORPORATION
Priority to JP61094396A priority patent/JPS61293919A/ja
Assigned to REPLIGEN CORPORATION A CORP. OF DELAWARE reassignment REPLIGEN CORPORATION A CORP. OF DELAWARE ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: ABBOTT BIOTECH, INC. A CORP. OF DELAWARE
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/128—Chemically defined matrices for immobilising, holding or storing living parts, e.g. alginate gels; Chemically altering living parts, e.g. by cross-linking
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/022—Artificial gland structures using bioreactors
    • A—HUMAN NECESSITIES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/08—Simple coacervation, i.e. addition of highly hydrophilic material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • B01J13/16—Interfacial polymerisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/32—Polylysine, polyornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • This invention relates to a process for encapsulating tissue or individual cells so that they remain viable and in a protected state within a membrane which is permeable to nutrients, ions, oxygen, and other materials needed to both maintain the tissue and support its normal metabolic functions, but impermeable to bacteria, lymphocytes, and large proteins of the type responsible for immuochemical reactions resulting in rejection.
  • the process enables the production of, for example, an insulin producing system or other hormone producing system as it allows encapsulation of mammalian pancreatic beta cells, alpha cells, intact islets of Langerhans, and other tissues or tissue fractions which secrete hormones.
  • the capsules may be suspended in a culture medium and will excrete hormone over an extended period.
  • the capsules may also be used as an artificial pancreas which can be implanted, e.g., by injection, into a diabetic mammal and will function in vivo to excrete insulin and other hormones in response to ambient sugar concentration.
  • Encapsulated live cells, organelles, or tissue have many potential uses.
  • the encapsulated living material can be preserved in a permanent sterile environment and can be shielded from direct contact with large, potentially destructive molecular species, yet will allow free passage of lower molecular weight tissue nutrients and metabolic products.
  • the development of such an encapsulation technique could lead to systems for producing useful hormones such as insulin.
  • the mammalian tissue responsible for the production of the material would be encapsulated in a manner to allow free passage of nutrients and metabolic products across the membrane, yet prohibit the passage of bacteria.
  • membrane permeability could be controlled, it is possible that this approach could also lead to artificial organs which could be implanted in a mammalian body, e.g., a diabetic, without rejection and with controlled hormone release, e.g., insulin release triggered by glucose concentration.
  • the instant invention provides a method of encapsulating core material such as living tissue, individual cells, or biologically active materials in a semipermeable membrane.
  • the basic approach involves suspending the tissue to be encapsulated in a physiologically compatible medium containing a water soluble substance that can be made insoluble in water, that is, gelled, to provide a temporary protective environment for the tissue.
  • the medium is next formed into droplets containing the tissue and gelled, for example, by changing conditions of temperature, pH, or ionic environment.
  • the "temporary capsules" thereby produced are then subjected to a treatment, which can be a known treatment, that results in the production of membranes of a controlled permeability (including impermeability) about the shape-retaining temporary capsules.
  • the temporary capsules can be fabricated from any nontoxic, water soluble substance that can be gelled to form a shape retaining mass by a change of conditions in the medium in which it is placed, and also comprises plural groups which are readily ionized to form anionic or cationic groups. The presence of such groups in the polymer enables surface layers of the capsule to be cross-linked to produce a "permanent" membrane when exposed to polymers containing multiple functionalities of the opposite charge.
  • the presently preferred material for forming the temporary capsules is polysaccharide gums, either natural or synthetic, of the type which can be (a) gelled to form a shape retaining mass by being exposed to a change in conditions such as a pH change or by being exposed to multivalent cations such as Ca++; and (b) permanently "crosslinked” or hardened by polymers containing reactive groups such as amine or imine groups which can react with acidic polysaccharide constituents.
  • the presently preferred gum is alkali metal alginate.
  • Other water soluble gums which may be used include guar gum, gum arabic, carrageenan, pectin, tragacanth gum, xanthan gum or acidic fractions thereof.
  • gelatin or agar may be used in place of the gums.
  • the preferred method of formation of the droplets is to force the gum-nutrient-tissue suspension through a vibrating capillary tube placed within the center of the vortex created by rapidly stirring a solution of a multivalent cation. Droplets ejected from the tip of the capillary immediately contact the solution and gel as spheroidal shaped bodies.
  • the preferred method of forming a permanent semipermeable membrane about the temporary capsules is to "crosslink" surface layers of a gelled gum of the type having free acid groups with polymers containing acid reactive groups such as amine or imine groups. This is typically done in a dilute solution of the selected polymer. Generally, the lower the molecular weight of the polymer, the greater the penetration into the surface of the temporary capsule, and the greater the penetration, the less permeable the resulting membrane. Permanent crosslinks are produced as a consequence of salt formation between the acid reactive groups of the crosslinking polymer and the acid groups of the polysaccharide gum. Within limits, semipermeability can be controlled by setting the molecular weight of the crosslinking polymer, its concentration, and the duration of reaction.
  • Crosslinking polymers which have been used with success include polyethylenimine and polylysine. Molecular weight can vary, depending on the degree of permeability required, between about 3,000 and 100,000 or more. Good results have been obtained using polymers having an average molecular weight on the order of 35,000.
  • the capsules can be engineered to have a selected in vivo useful life by astute selection of the cross-linking polymer.
  • Proteins or polypeptide crosslinkers e.g., polylysine
  • Cross-linkers not readily digestible in mammalian bodies, e.g., polyethyleneimine, result in longer lasting membranes.
  • the temporary capsules it is possible to improve mass transfer within the capsule after formation of the permanent membrane by re-establishing the conditions under which the material is liquid, e.g., removing the multivalent cation. This can be done by ion exchange, e.g., immersion in phosphate buffered saline or citrate buffer. In some situations, such as where it is desired to preserve the encapsulated tissue, or where the temporary gelled capsule is permeable, it may be preferable to leave the encapsulated gum in the crosslinked, gelled state.
  • An alternative method of membrane formation involves an interfacial polycondensation of polyaddition similar to the procedure disclosed in U.S. application Ser. No. 606,166.
  • the instant invention provides a tissue implantation method which does not require surgery and which overcomes many of the problems of immune rejection.
  • the capsules are injected into a suitable site in a mammalian body, and function normally until the tissue expires, or until natural body processes succeed in isolating the capsules so that substances required for viability of the tissue are no longer available.
  • the mammalian body may accordingly be provided with the specialized function of the tissue as long as desired.
  • mammalian Islets of Langerhans, or islet preparations containing selected amounts of alpha, beta, and/or delta cells from islets are encapsulated in polylysine and polyethyleneimine cross-linked alginate membranes. These may be periodically injected, e.g., into the peritoneal cavity of a diabetic mammalian body and function as an artificial pancreas.
  • Other objects of the invention include the provision of encapsulated living tissue useful for producing hormones such as insulin and for effecting complex chemical changes characteristic of the in vivo tissue, to provide an insulin generation system, to provide a body fluid detoxifying system, and to provide encapsulated activated charcoal.
  • Another object of the invention is to provide a method of implanting living tissue in mammalian bodies and to provide a non-surgical tissue implantation technique. Still another object is to provide a method of encapsulating living tissue which allows the production of capsules having a high surface area to volume ratio and membranes with a preselected in vivo residence time. Another object of the invention is to provide an artificial pancreas.
  • FIGURE of the drawing schematically illustrates a preferred method of encapsulating living tissue suitable for use in the process of the invention, and the product microcapsule.
  • the tissue, organelle, or cell to be encapsulated is prepared in accordance with well-known prior art techniques in finely divided form and suspended in an aqueous medium suitable for maintenance and for supporting the ongoing metabolic processes of the particular tissue involved. Media suitable for this purpose are available commercially.
  • the average diameter of the material to be encapsulated can vary widely between less than a micron to several millimeters. Mammalian islets of Langerhans are typically 140 to 200 microns in diameter. Of course, individual cells such as pancreatic beta cells, alpha cells, delta cells, or various ratios thereof, whole islet of Langerhans, individual hepatocytes, organelles, or other tissue units may be encapsulated as desired. Also, microorganisms may be encapsulated as well as non-living materials such as biological materials.
  • the solution is then formed into droplets containing tissue together with its maintenance medium and is immediately rendered water insoluble and gelled, at least in a surface layer.
  • the shape-retaining temporary capsules are provided with a permanent semipermeable membrane.
  • the capsule interior may be reliquified after formation of the permanent membrane. This is done by re-establishing the conditions in the medium at which the material is soluble.
  • the material used to form the temporary capsules may be any non-toxic, water-soluble material which, by a change in the surrounding temperature, pH, or ionic environment or concentration, can be converted to a shape retaining mass.
  • the material also contains plural, easily ionized groups, e.g., carboxyl or amino groups, which can react by salt formation with polymers containing plural groups which ionize to form species of opposite charge.
  • this type of material enables the deposition of a permanent membrane of a selected porosity and a selected in vivo lifespan in surface layers of the temporary capsule.
  • the presently preferred materials for forming the temporary capsule are water-soluble, natural or synthetic polysaccharide gums. Many such materials are commercially available. They are typically extracted from vegetable matter and are often used as additives to various foods. Sodium alginate is the presently preferred water soluble gum. Other usable gums include guar gum, gum arabic, charageenan, pectin, tragacanth gum, xanthan gum, or their acidic fractions.
  • These materials comprise glycoside-linked saccharide chains. Many contain free acid groups, which are often present in the alkali metal ion form, e.g., sodium form.
  • a multivalent ion such as calcium or strontium is exchanged for the alkali metal ion, the liquid, water-soluble polysaccharide molecules are "crosslinked" to form a water insoluble, shape-retaining gel which can be resolubilized on removal of the ions by ion exchange or via a sequestering agent. While essentially any multivalent ion which can form a salt is operable, it is preferred that physiologically compatible ions, e.g., calcium, be employed. This tends to preserve the tissue in the living state. Other multivalent cations can be used for less fragile material.
  • gums can be switched between the water soluble and gelled, water insoluble state simply by changing the pH of the medium in which they are dissolved.
  • a typical tissue-tissue medium-gum solution composition comprises equal volumes of tissue in its medium and a one to two percent solution of gum in physiological saline.
  • a 1.0 to 1.5 percent solution has been used with success.
  • gelatin or agar When encapsulating materials which can resist changes in temperature, gelatin or agar may be used to form the temporary capsules. These can be gelled by injection into a low temperature environment. Other water soluble substances such as hydroxyethyl methacrylate may also be used.
  • step BC of the drawing A beaker 10 containing an aqueous solution of multivalent cation, e.g., 1.5 percent CaCl 2 solution, is fitted with a magnetic stirring bar 11 and stirrer 12. The stirring mechanism is actuated to produce a vortex 14 having a hollow center 16. A capillary tube 18 of a selected inside diameter is positioned within hollow region 16 of the vortex and fitted with a vibrator 20.
  • multivalent cation e.g. 1.5 percent CaCl 2 solution
  • the suspension containing tissue and the solubilized gum is fed through the capillary.
  • the effect of surface tension which would induce the formation of relatively large droplets is minimized by the vibrator so that droplets, illustrated at 22, of a size comparable to the inside diameter of the capillary, are shaken off of the capillary tip. These immediately contact the solution where they absorb calcium ions. This results in "crosslinking" of the gel and in the formation of a shape-retaining, high viscosity protective temporary capsule containing the suspended tissue and its medium.
  • the capsules collect in the solution as a separate phase and are separated by aspiration.
  • a small amount of polymer of the type used for permanently crosslinking the gum is included in the solution together with the multivalent ions (or other solution capable of gelling the particular gum employed). This results in the formation of permanent crosslinks. Capsules of this type have certain advantages if the goal is to preserve the tissue.
  • a semipermeable membrane is deposited about the surface of the temporary capsules.
  • interfacial polymerization techniques can be exploited.
  • interfacial polymerization a pair of at least difunctional mutually reactive monomers, or a monomer and a relatively low molecular weight polymer, one of which is soluble in polar solents such as water and the other of which is soluble in hydrophobic solvents such as hexane, are caused to react at the interface of an emulsion of the water-in-oil type.
  • the material to be encapsulated is suspended or dissolved in water together with the water soluble component of the reaction, the aqueous phase is emulsified in a hydrophobic solvent, and the complementary monomer is added to the continuous phase of the system so that polymerization occurs about the aqueous droplets.
  • This technique may be used in accordance with the instant invention if the water soluble reactant is dissolved in an aqueous solution, and the solution is used to suspend the temporary capsules.
  • This liquid suspension is then emulsified in, for example, hexane, or a hexane-chloroform mix.
  • the complementary monomer is next added, preferably incrementally, to induce interfacial polymerization at the surface of the aqueous droplets. Because of the gelled mass of polysaccharide surrounding the suspended tissue, and especially if suitably buffered polyfunctional amino-group containing polymers such as certain proteins are employed as the water-soluble reactant, the process is such that the tissue survives the encapsulation in a healthy condition.
  • the substances useful in forming membranes with the polyfunctional amines include diacids, diacid halides, and multifunctional sulfonyl halides.
  • diamines, polyols, and diols may be used. Molecules containing plural amine groups may also be crosslinked with glutaraldehyde to form a membrane.
  • Another useful method of membrane formation is by interfacial polymerization utilizing polyaddition reactions. In this case, for example, multifunctional amines absorbed in surface layers of the temporary capsules are reacted with epichlorohydrin, epoxidized polyesters, or diisocyanate.
  • the preferred method of forming the membrane is to permanently cross link surface layers of the droplets by subjecting them to an aqueous solution of a polymer containing groups reactive with functionalities in the gel molecules.
  • a polymer containing groups reactive with functionalities in the gel molecules Certain long chain quaternary ammonium salts may be used for this purpose in some circumstances.
  • polymers containing acid reactive groups such as polyethylenimine and polylysine may be used. In this situation, the polysaccharides are crosslinked by interaction between the carboxyl groups and the amine groups.
  • permeability can be controlled by selecting the molecular weight of the crosslinking polymer used.
  • a solution of polymer having a low molecular weight, in a given time period, will penetrate further into the temporary capsules then will a high molecular weight polymer.
  • the degree of penetration of the crosslinker has been correlated with the resulting permeability. In general, the higher the molecular weight and the less penetration, the larger the pore size.
  • polymers within the molecular weight range of 3,000 to 100,000 daltons or greater may be used, depending on the duration of the reaction, the concentration of the polymer solution, and the degree of permeability desired.
  • reaction conditions using polylysine of average molecular weight of about 35,000 daltons, involved reaction for two minutes, with stirring, of a physiological saline solution containing 0.0167 percent polylysine.
  • Optimal reaction conditions suitable for controlling permeability in a given system can readily be determined empirically without the excercise of invention.
  • the permanent capsule membrane comprises polysaccharide (a readily injestible substance), cross-linked with either or both a polypeptide or protein, e.g., polylysine, or a synthetic substance, e.g., polyethyleneimine.
  • Polymers vary with respect to the rate at which they can be dispersed in vivo. Some are digested without difficulty, e.g., protein; others are slowly degraded, and still others remain indefinitely.
  • the process of the invention contemplates cross-linking with one or more polymers to produce capsules having a selected rate of dissolution in vivo, ranging generally between a few hours or days to substantial permanence.
  • the example which follows discloses how to produce capsules which remain intact at least about two months within the peritoneal cavity of rats.
  • the invention is not limited to these particular capsule membranes nor to capsules of this degree of in vivo life.
  • the optimal in vivo life of the microcapsules depends upon the their intended use and their site of implantation. Those skilled in the art will be able to produce microcapsules of a selected in vivo lifespan empirically without the exercise of invention in view of this disclosure.
  • capsules may be collected which comprise a permanent semipermeable membrane surrounding a gelled solution of gum, tissue compatible culture medium, and tissue particles. If the object is simply to preserve the tissue in a protective environment, no further steps need be done. However, if mass transfer is to be promoted within the capsules and across the membranes, it is preferred to reliquify the gel to its water soluble form. This may be done by reestablishing the conditions under which the gum is a liquid, e.g., changing the pH of the medium or removing the calcium or other multifunctional cations used.
  • the medium in the capsule can be resolubilized simply by immersing the capsules in phosphate buffered saline, which contains alkali metal ions and hydrogen ions.
  • Monovalent ions exchange with the calcium or other multifunctional ions within the gum when, as shown at stage E of the drawing, the capsules are immersed in the solution with stirring.
  • Other salts e.g. sodium citrate, may be used for the same purpose.
  • the capsules may be desirable to treat the capsules so as to tie up free amino groups or the like which would otherwise impart to the capsules a tendency to clump. This can be done, for example, by immersing the capsules in a solution of sodium alginate.
  • the invention contemplates the injection of encapsulated, finely divided tissue, multicellular fractions thereof, or individual cells into an appropriate site within a mammalian body for the purpose of providing the body, at least temporarily, with the tissue's specialized physiological function.
  • the procedure has the dual advantages of obviating the need for surgical implantation (although capsules may be implanted surgically if desired) and successfully dealing with the problems of immune rejection and natural physical isolation.
  • the capsule membranes consist of substances which are injested after expiration of the tissue. As noted above, this can be accomplished by employing a cross-linker which resists in vivo breakdown so that a given useful in vivo life is attained.
  • Islets of Langerhans were obtained from rat pancreas and added to a complete tissue culture (CMRL-1969 Connaught Laboratories, Toronto, Canada) at a concentration of approximately 10 3 islets per milliliter.
  • the tissue culture contains all nutrients needed for continued viability of the islets as well as the amino acids employed by the Beta cells for making insulin.
  • Four-tenths of a milliliter of the islet suspension was then added to a one-half milliliter volume of 1.2 percent sodium alginate (Sigma Chemical Company) in physiological saline.
  • the capsules resist clumping, and many can be seen to contain islets of Langerhans.
  • these capsules Under the microscope, these capsules have an appearance illustrated in the drawing. They comprise a very thin membrane 24 which encircle an islet 26 within which individual cells 28 can be seen. Molecules having a molecular weight up to about 100 thousand can traverse membrane 24. This allows oxygen, amino acids, nutrients, and plasma components used in culture media (e.g., fetal calf plasma components) to reach the islet and allows insulin to be excreted.
  • culture media e.g., fetal calf plasma components
  • microcapsules made in accordance with Example 1 containing approximately 15 islets were suspended in 3 milliliters of CMRL-1969.
  • CMRL-1969 a group consisting of CMRL--1969.
  • the capsules excreted, in one run, 67 microunits/ml insulin in 1.5 hours.
  • 68 microunits/ml insulin were produced in the same amount of time.
  • Diabetic rats with blood gluclose levels in the range of 500-700 mg/dl were each treated with approximately 10 3 islets encapsulated as set forth in Example 1, and suspended in physiological saline.
  • the capsules were introduced by injection into the peritoneal cavity using a number 19 needle fitted to a syringe. Blood sugar levels were assayed daily and uniformly found to be below 300 mg/dl. Animals sacrificed after two months showed no signs of toxic reaction about the site of the implantation. Capsules removed from sacrificed animals after a two-month in vivo life were intact and showed no signs of degredation.
  • liver tissue in vitro, can ingest toxins from its environment. Accordingly, toxins of a molecular weight low enough to pass through the semipermeable membranes are injested and destroyed by the tissue. Essentially all toxins treated by the liver are low molecular weight materials. However, the toxins may be protein-complexed. Capsular permeability can be varied according to the need.
  • example 1 The procedure of example 1 is repeated except that particulate activated charcoal is suspended directly in the sodium alginate solution, the half milliliter of tissue suspension is omitted, and polylysine of an average molecular weight of 35,000 is used as a crosslinker.
  • the charcoal particles are smaller than the smallest inside diameter of the capillary used to produce the droplets, charcoal of high surface area surrounded by a semipermeable membrane results. These effectively prohibit the escape of charcoal chips or dust, yet can be used to absorb medium range molecular weight materials (up to about 2,000 daltons) from fluid passed about the capsules.

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Priority Applications (19)

Application Number Priority Date Filing Date Title
US06/024,600 US4352883A (en) 1979-03-28 1979-03-28 Encapsulation of biological material
GB8008971A GB2046209B (en) 1979-03-28 1980-03-17 Encapsulation of chemically active biologically active materials and viable tissue
CA000348524A CA1145258A (en) 1979-03-28 1980-03-26 Encapsulation of viable tissue and tissue implantation method
DK130580A DK130580A (da) 1979-03-28 1980-03-26 Indkapsling af levedygtigt vaev og fremgangsmaade til implantation af vaev
IT67472/80A IT1133081B (it) 1979-03-28 1980-03-27 Procedimento per incapsulare ed impiantare un tessuto vivente
SE8002357A SE448060B (sv) 1979-03-28 1980-03-27 Forfarande for inkapsling av ett kernmaterial inom ett selektivt permeabelt membran samt kapsel
FR8006875A FR2452285B1 (fr) 1979-03-28 1980-03-27 Procede d'encapsulation d'un tissu viable ou d'une matiere biologiquement active a implanter, et capsules obtenues
BE0/199986A BE882476A (fr) 1979-03-28 1980-03-27 Procede d'encapsulation d'un tissu viable ou d?une matiere biologiquement active a implanter et capsules obtenues
CH2423/80A CH653914A5 (de) 1979-03-28 1980-03-27 Verfahren zum einkapseln eines chemisch aktiven kernmaterials.
CH187/84A CH657786A5 (de) 1979-03-28 1980-03-27 Verfahren zum einkapseln eines kernmaterials innerhalb einer semipermeablen membran.
DE19803012233 DE3012233A1 (de) 1979-03-28 1980-03-28 Verfahren zum einkapseln von lebensfaehigem gewebe in eine membran
JP3912380A JPS55157502A (en) 1979-03-28 1980-03-28 Live tissue encapsulation and tissue transplantation
NO800901A NO160380C (no) 1979-03-28 1980-03-28 Fremgangsmaate for innkapsling av et kjernemateriale, f.eks. levedyktig vev, i en membran.
FR8020768A FR2457688B1 (fr) 1979-03-28 1980-09-26 Organe artificiel, sous forme de microcapsules, convenant pour son implantation dans le corps d'un mammifere
US06/259,595 US4391909A (en) 1979-03-28 1981-05-01 Microcapsules containing viable tissue cells
US06/372,835 US4409331A (en) 1979-03-28 1982-04-28 Preparation of substances with encapsulated cells
GB08227739A GB2119734B (en) 1979-03-28 1982-09-29 Encapsulated living tissue
GB08306682A GB2119737B (en) 1979-03-28 1983-03-11 Encapsulation of viable tissue
JP61094396A JPS61293919A (ja) 1979-03-28 1986-04-23 化学的に活性な材料のカプセル化法

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JP (2) JPS55157502A (cs)
BE (1) BE882476A (cs)
CA (1) CA1145258A (cs)
CH (2) CH657786A5 (cs)
DE (1) DE3012233A1 (cs)
DK (1) DK130580A (cs)
FR (2) FR2452285B1 (cs)
GB (1) GB2046209B (cs)
IT (1) IT1133081B (cs)
NO (1) NO160380C (cs)
SE (1) SE448060B (cs)

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BE882476A (fr) 1980-09-29
CH653914A5 (de) 1986-01-31
FR2452285A1 (fr) 1980-10-24
NO160380B (no) 1989-01-02
SE8002357L (sv) 1980-09-29
IT8067472A0 (it) 1980-03-27
JPS6239131B2 (cs) 1987-08-21
JPS61293919A (ja) 1986-12-24
DE3012233C2 (cs) 1988-03-31
DK130580A (da) 1980-09-29
CA1145258A (en) 1983-04-26
FR2457688B1 (fr) 1986-12-05
FR2457688A1 (fr) 1980-12-26
JPS6242889B2 (cs) 1987-09-10
NO800901L (no) 1980-09-29
IT1133081B (it) 1986-07-09
DE3012233A1 (de) 1980-11-20
SE448060B (sv) 1987-01-19
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GB2046209B (en) 1983-09-14
NO160380C (no) 1989-04-19
GB2046209A (en) 1980-11-12
FR2452285B1 (fr) 1986-12-05

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